Single-molecule profiling of tau aggregates in tauopathies: From test tube to patient

In recent decades, single-molecule methods have transformed the physical sciences, offering unprecedented insights into the complexity of biological systems at molecular resolution. In particular, fluorescence-based methods now enable the detection and imaging of individual biomolecules and their interactions below the diffraction limit (< 200 nm). Despite their potential, these techniques remain underutilised in biomedical research, especially in complex biological samples where molecular targets are present at very low concentrations. A notable example are tauopathies, a group of neurodegenerative diseases in which pathological aggregation of the tau protein plays a central role. Current approaches often fail to resolve the small, early-stage tau aggregates that are believed to be a major neurotoxic species. In this thesis, I address this gap by developing and applying two complementary single-molecule platforms: MAPTau, a super-resolution Single-Molecule Pull-Down assay, and Single-Molecule Array (Simoa). Together, these tools enable the ultrasensitive detection and detailed characterisation of tau aggregates in brain and biofluid samples. Beyond quantification, they allow detailed profiling of aggregate morphology, post-translational modifications, and protein–protein interactions at the single-aggregate level. I demonstrate how these approaches distinguish tau aggregate properties across Alzheimer’s disease, primary tauopathies, and healthy controls, revealing disease-specific differences in abundance, phosphorylation, and seeding activity. To map how tau’s interactome evolves with aggregation I combine single-molecule assays with proteomics. Finally, I explore tau aggregate heterogeneity across Braak stages, investigate early tau aggregation in Down syndrome organoids, and identify molecular features associated with tau bioactivity. Together, these studies provide new molecular insights into tau aggregation and its role in neurodegeneration. They establish single-molecule assays as powerful tools to study tau pathology in human tissue and translational models. By resolving previously inaccessible features of tau aggregates, this work lays the foundation for improved diagnostics and mechanistic understanding in Alzheimer’s disease and related tauopathies.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Böken, Dorothea

Advisor dc:contributor.advisor

• Klenerman, David

• Tau
• Single-molecule
• Tauopathies
• Super-resolution

Licence

• https://www.repository.cam.ac.uk/bitstreams/7429f09f-925f-466f-aeb6-2c35c16ee03e/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.121423

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/389599